Drug abuse involves the pharmacologic activation of neural pathways involved in reward. Dopamine mediates these pathways and drugs of abuse bypass the normal stimuli for reward by increasing dopamine release more directly. This non-physiological activation then produces changes in synaptic transmission that presumably underlie tolerance, physical dependence and drug craving. Drug abuse therefore provides a model for neural plasticity in the reward pathway that has clear relevance for behavior. The long-term goal of this proposal is to understand how drugs of abuse alter synaptic transmission. The strategy is to focus on the membrane trafficking of proteins involved in neurotransmitter release and signal transduction, their regulation and the physiological significance of this regulation for the release of dopamine. Dr. Sulzer will use direct, amperometric methods to study the kinetic features of quantal dopamine release and in continued collaboration with Dr. Edwards, the role of vesicular monoamine transporter 2 (VMAT2) and D2 autoreceptors in the regulation of quantal size and the readily releasable pool of synaptic vesicles. To assess the potential for presynaptic regulation of quantal size, Dr. Edwards will study the membrane trafficking of VMAT2. Previous work has demonstrated the phosphorylation of V MAT2 and the closely related vesicular ACh transporter, and he will now address the role of two particular motifs in the trafficking of these proteins to large dense core vesicles (LDCVs) as well as synaptic vesicles, extend the analysis to primary neuronal culture and dopamine release with Dr. Sulzer, and use the new information about sorting signals to characterize the cytosolic sorting machinery. Dr. Kelly will explore two pathways involved in synaptic vesicle formation, using both pharmacologic and genetic approaches. In particular, he will examine the role of a brefeldin A-sensitive pathway in the recycling of proteins derived from LDCVs (such as VMAT2 and VAChT), and use mocha mice deficient in the adaptor protein AP-3 to assess the physiological significance of this pathway in collaboration with Dr. Sulzer. Dr. von Zastrow will address the role of membrane trafficking in the regulation of opiate receptors, and dissect the mechanisms responsible for their internalization after activation by ligand. In addition, he will characterize trafficking of the delta-opiate receptor to LDCVs in collaboration with the Edwards and Kelly groups. He will also test using knock-in mice the novel hypothesis that morphine's susceptibility to abuse derives from its failure to induce mu-opiate receptor internalization by altering the sorting sequences in vivo.